Iontophoresis of inositol 1, 4, 5-trisphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1,4-bisphosphate also triggered these events, but only at doses -100-fold higher, whereas no level of fructose-I, 6-bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5-trisphosphate triggered a Ca 2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985r J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca 2÷ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca 2÷ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca 2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca 2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca 2+ mobilization by sperm-egg interaction.A transient increase in intracellular free Ca 2÷ concentration ([Ca2*]i) ~ accompanies fertilization in both invertebrate (8, 9, 23) and vertebrate eggs (4,6,16,21), and considerable evidence suggests that such Ca 2+ pulses provide the primary regulatory signal that triggers the initiation of development in the zygote (13,18). The means by which sperm-egg interaction triggers intracellular Ca 2+ release in the egg is unknown, but Ca2+-induced Ca 2÷ release has been suggested to be involved (18,22).Recently, considerable evidence has accumulated that implicates the turnover of inositol lipids in the mobilization of intracellular Ca 2÷ seen in a variety of cell types during hormonal stimulation. In response to hormones such as acetylcholine, vasopressin, or platelet-derived growth factor, responsive cells such as pancreatic acinar cells, hepatocytes, or cultured fibroblasts, respectively, undergo a pronounced increase in the hydrolysis of the plasma membrane lipid, phosphatidylinositol-4, 5-bisphosphate (PIP2), releasing into the cytosol the water-soluble product, inositol-1, 4, 5-trisphosphate (IP3), which, in turn, can trigger Ca z÷ release from intracellular stores in these and other cells (see references 1 t Abbrevmtions used in this paper.[Ca2÷]i, intracellular free Ca 2÷ concentration; IP2, inositol-l, 4-bisphosphate; IP~, inositol-l, 4, 5-trisphosphate; pCa, negative log of free Ca 2+ concentration; PIP2, phosphatidylinositol-4, 5-bisphosphate. and 2 for review). That hydrolysis of PIP2, yielding IP3, may also be the trigger for Ca 2÷ release in eggs at fertilization is suggested by recent observations on sea urchin eggs. Fertilization of these eggs triggers a rapid turnover of PIP2 (24), and microinjection of IP3 into the unfert...
1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycero1 (OcozGro) and 1 -oleoyl-2-acetyl-glycerol (OleAcGro) on agonist-induced platelet activation processes were compared with those of the phorbol ester, phorbol12-myristate 13-acetate (PMA), using appropriately labelled washed human platelets.2. Pre-treatment (10 -300 s) with OcozGro (1 5 -60 pM) or PMA (16 nM) before addition of thrombin (0.2 U/ ml) or, addition of these agents 10-20 s after thrombin, resulted in a significant reduction (20-80%) in the extent of thrombin-induced intracellular CaZ ' ([Ca2'Ii) mobilisation and arachidonate/thromboxane B2 release.OleAcGro (62-125 pM) had no effect on thrombin-induced [Ca2+Ii elevations but had a slight (15%) inhibitory effect on thrombin-induced arachidonate release with a 5-min pre-incubation. Addition of OcozGro, PMA orOleAcGro on their own caused no rise in [Ca2 'Ii levels or arachidonate release.3. Collagen (20 pg/ml) induced substantial arachidonate release without a detectable rise in [Ca2+Ii. Pretreatment (10-300 s) with OcozGro (15-60 pM), PMA (16 nM) or OleAcGro (62 pM) before collagen addition or addition of these agents 30 -60 s after collagen addition resulted in a significant potentiation of arachidonate release (1.2 -2-fold over control), even though thromboxane B2 formation in response to collagen was inhibited in the presence of OcozGro or PMA.4. Both Oco2Gro and PMA had dual effects on 5-hydroxytryptamine secretion induced by thrombin or collagen. Short pre-incubations ( < 2 min) with these agents caused a potentiation of sub-maximal agonist-induced secretion, while not affecting secretion induced by maximal agonist concentrations. With longer pre-incubation times (5 -15 min) however, a significant reduction in the level of agonist-induced secretion in the presence of Oco2Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 pM), suggesting that inhibition of thromboxane B2 formation alone does not account for inhibition of 5-hydroxytryptamine secretion. OleAcGro had no inhibitory effects on agonist-induced secretion even though it potentiated it (with < 2-min incubations) at sub-maximal agonist concentrations.5. Time courses of phosphorylation of a 45-kDa protein, a marker of protein kinase C activation, in 32P-labelled platelets showed that while OcozGro (60 pM) and PMA (16 nM) caused a 4-5-fold increase in 32P-labelling of this protein over a 5-min incubation period, OleAcGro (62-125 pM) caused a 1.5-fold increase in labelling which was only maintained for a 10-30-s period. The inability of OleAcGro to exert any significant inhibitory effects on agonist-induced platelet responses may therefore be due to insufficient activation of protein kinase C and/or phosphorylation of the 45-kDa protein. Hence, OcozGro may be a better tool as a diacylglycerol analogue. However, the potentiatory effects of OleAcGro with short pre-incubations (agonist-induced 5-hydroxytryptamine secretion and collagen-induced arachidonat...
Elevation in cell cAMP content can inhibit mitogenic signaling in cultured human airway smooth muscle (HASM) cells. We studied the effects of the type 3-selective phosphodiesterase inhibitor siguazodan, the type 4-selective phosphodiesterase inhibitor rolipram, and the nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) on proliferation of cultured HASM cells. At concentrations selective for the type 3 phosphodiesterase isoform, siguazodan inhibited both [3H]thymidine incorporation (IC50 2 μM) and the increase in cell number (10 μM; 64% reduction) induced by platelet-derived growth factor-BB (20 ng/ml). These effects were mimicked by IBMX. At concentrations selective for type 4 phosphodiesterase inhibition, rolipram was without effect. A 20-min exposure to siguazodan and rolipram did not increase whole cell cAMP levels. However, in HASM cells transfected with a cAMP-responsive luciferase reporter (p6CRE/Luc), increases in cAMP-driven luciferase expression were seen with siguazodan (3.9-fold) and IBMX (16.5-fold). These data suggest that inhibition of the type 3 phosphodiesterase isoform present in airway smooth muscle results in inhibition of mitogenic signaling, possibly through an increase in cAMP-driven gene expression.
1 Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nM) and endothelium-derived relaxing factor (EDRF, NO; K., = 0.7 nM) in a concentrationdependent manner when exposed to bradykinin.
It has previously been reported that murine macrophages can respond chemotactically and mitogenically to the serine proteinase thrombin. There is a similar response in these macrophages to catalytically inactivated thrombin or to peptide fragments of the thrombin B-chain [Bar-Shavit, Kahn, Mann and Wilner (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 976-980]. However, the existence of a non-proteolytic mechanism of thrombin receptor activation in mononuclear cells was not evident in the present study using U937 human monocytic cells. The ability of thrombin to stimulate intracellular Ca2+ mobilization, actin polymerization or cell proliferation was not mimicked by N alpha-tosyl-L-lysine chloromethyl ketone (TLCK)-treated thrombin or by a synthetic 14-amino-acid peptide (single amino acid letter code YPPWNKNFTENDLL) corresponding to a part of the B-chain of thrombin which was reported to be mitogenic in murine macrophages. Evidence was obtained, however, in U937 cells for the presence of proteolytic-dependent thrombin receptor similar to the thrombin receptor expressed in platelets, which following thrombin cleavage exposes a new N-terminal tethered ligand. In support of this, a thrombin-receptor-derived hexapeptide (TRP; sequence SFLLRN), corresponding to a part of the thrombin receptor tethered ligand, mimicked all the actions of thrombin in U937 cells. Further, TRP and thrombin cross-desensitized U937 cells to subsequent stimulation with either TRP or thrombin, suggesting that TRP acted through the same U937 cell surface receptor as did thrombin. Thrombin activation of U937 monocytic cells can therefore be accounted for entirely by a proteolytic mechanism of thrombin receptor activation.
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